Glycosylated proteins (e.g. glycosylated albumin) in blood consist of glucose and blood protein (e.g. albumin) bonded by non-enzymatic covalent bonds. Aldehyde groups of glucose and amino groups of protein are responsible for the covalent bonds. Glycosylated albumin or the like in blood reflects the blood sugar level of a certain period in the past. Consequently, the measured value of glycosylated albumin has recently been noted as an important index for diagnosis and observation of conditions of diabetes.
Examples of the methods for measuring the amount of glycosylated proteins include high-performance liquid chromatography (HPLC) method, immunoassay, chemical method (NBT test), dye-binding methods, enzymatic methods, and the like. Above all, the enzymatic method employing an Amadori compound oxidoreductase such as fructosyl amino acid oxidase (FAOD) makes it possible to measure the amount of glycosylated proteins more accurately and promptly than the other methods.
The method for measuring the amount of glycosylated proteins by the use of FAOD is performed as follows. First, the glycosylated proteins are treated with protease so as to decompose them into peptides or amino acids, because FAOD acts with difficulty on large proteins or long peptides. Then, the decomposed products are treated with FAOD.
Thus, by measuring the amount of hydrogen peroxide produced by the treatment with FAOD by electric method or enzymatic method, the concentration of glycosylated protein can be determined.
As the enzymatic method for measuring the hydrogen peroxide, there is, for example, a method in which peroxidase (POD) and a substrate generating color by oxidation are used to determine the degree of color generation of the substrate.
In the method for measuring the amount of glycosylated proteins by the enzymatic method, there is a problem in that even if the glycosylated proteins are treated with protease beforehand, short peptides capable of being a substrate for an Amadori compound oxidoreductase cannot sufficiently be produced. In particular, protease acts only with difficulty on glycosylated proteins contained in blood. In order to avoid this problem, an improvement method has been suggested in which glycosylated proteins are treated with protease in the presence of POD before the treatment with the oxidoreductase (WO 96/34977). According to this improvement method, the short peptides capable of being the above-mentioned substrate can be produced effectively. Moreover, it is not clear why the glycosylated proteins are well decomposed if they coexist with POD during the treatment with protease.
However, this improvement method has the following problem. Since POD itself is a glycosylated protein and decomposed with protease to produce a substrate for the oxidoreductase, the results of the method are less reliable.